Apparatus and methods for detecting DNA in biological samples

ABSTRACT

Apparatus and methods are described for detecting target DNA in a biological sample using capture probes and electrically-assisted hybridization. The reaction cell is formed with an attachment surface of aluminum oxide for better thermal and physical properties, and the aluminum oxide surface is coated with anti-DIG antibody to provide a convenient attachment layer for the capture probes allowing their correct orientation, while the capture probes are formed with a DIG-label so that they attach to the surface of the cell through an anti-DIG/DIG linkage.

This is a Divisional of U.S. application Ser. No. 11/599,479, filed Nov.15, 2006 which is a Divisional of U.S. application Ser. No. 10/216,928,filed Aug. 13, 2002 (now U.S. Pat. No. 7,153,687, issued Dec. 26, 2006).

FIELD OF THE INVENTION

This invention relates to apparatus and methods for detecting DNA inbiological samples. In particular the present invention relates to novelapparatus for detecting DNA sequences using electrically-assistednucleic acid hybridization and to methods for optimizing the performanceof such apparatus.

BACKGROUND OF THE INVENTION

The use of electrically-assisted nucleic acid hybridization is a knowntechnique in the analysis of biological samples containing DNA, e.g.blood, plasma, urine etc. Conventionally, a chip for DNA detection isformed from one of a variety of materials including glass, silica andmetal. On the surface of the chip a number of electrical contacts areformed using known techniques. To detect a particular DNA sequence in abiological sample, capture probes consisting of complementary DNAfragments are attached to the chip surface. If a biological samplecontains the target DNA, the target DNA will bind to the complementaryDNA fragments by hybridization, and various imaging techniques may beused to detect such hybridization and thus the presence in the sample ofthe target DNA. By applying an electric current to the capture probes,the hybridization process may be accelerated and thus the detectionprocess is also accelerated. This basic hybridization technique isdescribed, for example, in U.S. Pat. No. 5,849,486.

PRIOR ART

In such devices and techniques, the major issues concerned are thebinding of the capture probes to the surface of the chip, and theimaging and visualization of the hybridization when it occurs.Concerning the latter, a traditional method of detection isfluorescence, but more recently Taton et el, (“Science” Vol. 289, 8 Sep.2000, pp 1757-1760) describes the use of streptavidin-coated goldnanoparticles for detection. In such a technique gold nanoparticles arecoated with an oligonucleotide sequence complementary to the target DNAsequence. A solution containing such coated gold nanoparticles is passedover the surface of the chip and gold particles will then bind to anyprobes to which the target DNA is bound. The gold particles themselvesare too small to be detected, but the chip surface may then be incubatedwith a solution containing silver ions. The silver ions are depositedonto the surface of the gold nanoparticles to form a visible silverlayer, which may be detected by conventional imaging apparatus.

Concerning the binding of the capture probes to the chip surface, onedifficulty is that the attachment of the DNA oligonucleotides onto achip surface (e.g. a silicon wafer) is a critical step that is highlysensitive to disturbances in experimental conditions. In particular whenan electric current is applied during the electrically assistedhybridization process, electrolysis may occur that can adversely affectsubsequent reactions and detection. It is known for example to coat thechip surface with an agarose gel and the DNA oligonucleotides used ascapture probes are labeled at one end with biotin and embedded in theagarose layer by virtue of the high affinity interaction between biotinand streptavidin that is dissolved in the agarose prior to the coatingof the chip surface.

However, there are a number of problems with using agarose. Firstly itis very difficult to manipulate a molten agarose gel so as to maintainan even coating on the chip. In addition the density and concentrationof the agarose gel must be precisely controlled. Thus manufacture of achip with a suitable agarose layer is very difficult. Furthermore onceapplied the gel must be kept moist to prevent drying and cracking, andagarose is very unstable and degrades at high temperatures, and so thechip must be refrigerated after application of the agarose layer. Theagarose layer is also very delicate and susceptible to mechanicalstresses, and thus the storage and transportation of the finished chipis difficult. In any event, the use of an agarose gel does not overcomethe potential problems caused by electrolysis.

SUMMARY OF THE INVENTION

According to the present invention there is provided apparatus fordetecting target DNA in a biological sample, comprising a substrateformed with at least one reaction cell, wherein said reaction cellincludes an aluminum oxide surface for the attachment of DNA captureprobes.

By means of this arrangement some of the problems with the prior art areovercome or at least mitigated because aluminum oxide provides a stableattachment surface that is easy to form and to manipulate, facilitatessubsequent handling and storage of the apparatus, and which overcomes orat least mitigates the problems with electrolysis.

The aluminum oxide attachment surface may be formed by oxidation of apreviously formed aluminum layer, or directly, for example bysputtering.

A method of attaching the capture probes to the aluminum oxide layerwould increase the convenience of manufacture. An attachment layeradsorbed to the aluminum oxide would perform this function. It is alsoimportant to be able to correctly orient the capture probes with respectto the attachment layer. A protein may perform both of these activities.Preferably this protein may be an antibody to a protein used to labelthe DNA capture probes, so that the capture probes may be linked to theattachment surface through an antibody-protein pair linkage in a definedorientation. Alternatively, however, the surface could be coated withany protein and the capture probes may be labeled with a protein havinga high affinity with the first protein, for example streptavidin and/orbiotin.

To reduce the background signal, the attachment surface may be coatedwith a reagent such as albumin, salmon sperm DNA, Ficoll, or otherprotein.

Viewed from another broad aspect the present invention includes a kitfor the detection of target DNA in a biological sample, said kitcomprising:

-   -   (a) a substrate including at least one reaction cell, said at        least one reaction cell having an aluminum oxide surface,    -   (b) a first reagent comprising capture probes including a        complementary DNA fragment to said target DNA,    -   (c) a buffer solution for receiving said sample,    -   (d) a second reagent comprising detection probes including a        complementary DNA fragment to said target DNA,    -   (e) a solution of streptavidin-coated gold nanoparticles, and        a solution containing silver ions.

Viewed from another aspect the present invention also extends to a DNAchip for detecting target DNA in a sample comprising a substrate havingan aluminum oxide surface, and a DNA capture probes attached to saidaluminum oxide surface.

Viewed from a still further aspect the invention also extends to amethod for detecting target DNA in a biological sample, comprising thesteps of:

-   -   (a) providing a reaction cell formed with an aluminum oxide        surface,    -   (b) coating said surface with a first protein to facilitate the        binding of the second protein that is attached to the capture        probe resulting in the correct orientation of said capture        probe,    -   (c) adding to said cell a solution containing capture probes        formed with a DNA sequence complementary to said target DNA and        labeled with a second protein having high affinity with said        first protein,    -   (d) supplying said sample to said cell,    -   (e) adding to said cell a solution containing target DNA        detection probes fowled with a DNA sequence complementary to        said target DNA,    -   (f) adding to said cell means for generating a detectable signal        in a cell where target DNA has been captured by said capture        probes and detected by said detection probes, and    -   (g) detecting said signal.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the present invention will now be described by wayof example and with reference to the accompanying drawings, in which:—

FIG. 1 is a schematic view illustrating a chip according to anembodiment of the invention and also illustrating the hybridization anddetection scheme,

FIGS. 2( a) to (d) show reaction cells obtained experimentally that showthat the presence of the capture probe enables the target DNA to bedetected,

FIGS. 3( a) to (d) show reaction cells obtained experimentally that showthat the presence of the target DNA is required before silver depositswill be formed,

FIGS. 4( a) to (d) show reaction cells obtained experimentally that showthat the intensity of the silver deposits increases with increasingconcentration of the target DNA in the sample, and

FIG. 5 shows reaction cells obtained experimentally that show that theintensity of the silver deposits varies with the concentration of thecapture probe.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Referring firstly to FIG. 1 there is shown schematically a novelapparatus for the detection of DNA in biological samples in accordancewith an embodiment of the present invention. The chip comprises asilicon wafer 1 on which a layer 2 of aluminum is fabricated, and alayer of aluminum oxide 3 is formed on the aluminum layer 2. Thealuminum oxide layer 3 is the attachment surface for the DNA probes aswill be described below.

The aluminum oxide layer 3 may be formed either by oxidization of thealuminum layer, or by direct deposition of alumina on the surface of thechip without the need for an aluminum layer at all. These possibilitieswill be now be described in more detail.

In a first method of forming the aluminum oxide layer, aluminum isallowed to grow on a clean aluminum surface that is exposed to oxygenand water. Silica chips that are fabricated with a layer of aluminum onthe surface are first rinsed with distilled water, cleaned by dippinginto 5% (w/v) NaOH solution for about 30 s, and then washed severaltimes with distilled water according to the technique described inSharma C P & Sunny M C “Albumin adsorption on to aluminum oxide andpolyurethane surfaces” Biomaterials, 1990; 11: 255-257. The chips arethen heated overnight at 37° C. in an oven with some water in acontainer to maintain moisture. Under these conditions the surface ofthe aluminum oxidizes to alumina with a thickness of about 50 Å. In amodification of this process the chips may be simply cleaned withdistilled water and then heated at 60° C. for 48 hours.

As an alternative to forming alumina by oxidation of an aluminum layer,alumina may be deposited directly on a silicon wafer according to thefollowing sputtering conditions:

Equipment: ARC-12M Sputtering system

RF power: 120 W

Base pressure: 1.04×10⁻⁵ torr

Process pressure: 5×10⁻³ torr

Gas flow: Ar/O₂=30.2/7.5 sccm

Stage rotation: 8 rpm

Sputter time: 45 minutes

Minimum thickness: 100 Å

Chip size: 5 mm×5 mm

The alumina-coated chip may be patterned by conventionalphotolithographic techniques. In addition, whichever method is used toform the alumina layer, prior to hybridization the coated chip is washedonce with 1×PBS (phosphate-buffered saline, pH 7.4) at room temperatureby pipetting the solution repeatedly over the chip surface. Of thealternative methods of forming the alumina layer, sputtering may bepreferred as studies show that is produces the lowest background signal.Whichever technique is used for forming the aluminum layer, thethickness may be at least 50 Å.

Aluminum oxide is preferred for the probe attachment surface becausecompared with, for example, agarose it is cheaper, more stable and moredurable. In addition aluminum oxide can be stored dry and at roomtemperature. Importantly, aluminum oxide also eliminates the problemsassociated with electrolysis. In addition, while agarose is difficult tohandle to form controlled layers, the pore size and thickness of thealumina layer can easily be controlled (for example by altering thesputtering conditions or the air moisture content). The aluminum oxidecan also easily be patterned using conventional photolithographictechniques. The use of a patterned aluminum oxide layer is advantageousbecause it can reduce the background signal due to reduced non-specificbinding, and by increasing the contrast between the area being imaged bythe detector and the background.

Referring back to FIG. 1 there is also shown schematically the basichybridization scheme using an apparatus and method according to anembodiment of the present invention. In particular, in FIG. 1 theattachment surface is a layer of aluminum oxide formed on aluminum. Acircular reaction well may be defined by depositing silicon oxide on thealuminum oxide attachment surface. Within each reaction well, captureprobes are attached to the aluminum oxide layer. The capture probes arenot attached directly, however, but through a linkage formed ofdigoxigenin (DIG) and anti-digoxigenin (anti-DIG) antibodies. Inparticular, anti-DIG antibodies are adsorbed by the aluminum oxidesurface, and the capture probes are formed with a DIG-label whereby thecapture probes may be linked via the anti-DIG antibodies to the aluminumoxide attachment surface. The aluminum oxide surface is very porous andhas the ability to bind many different molecules. The anti-DIG coatingfunctions as an attachment layer to ensure that the immobilized DNAcapture probes are correctly oriented. If no coating were applied, thereis a danger that the detection probe (to be described below) could bindto the aluminum oxide surface and cause interference in the detection oftarget DNA. In addition, if no coating were applied, the capture probemight bind to the aluminum oxide in the incorrect orientation. Inprinciple, the anti-DIG/DIG linkage could be replaced by any similarpair of compounds, e.g. an antibody and a target protein, or a pair ofproteins with very high affinity for each other, or any pair ofnon-protein molecules that are able to interact with each other. Itshould also be understood that the order of the antibody/protein orprotein/protein linkage could be reversed. For example while in thepreferred embodiment described herein anti-DIG is used as the reactivitylimiting coating and the capture probes are DIG-labeled this could bereversed. However, in practical terms it is easier to attach a smallmolecule such as DIG to a capture probe than it would be to attach anantibody to the capture probe. Furthermore it should be noted that thecapture probe could be directly attached to the aluminum oxide layer bythe addition of a terminal amine or aldehyde group.

It will also be understood that the capture probes are formed with a DNAsequence complementary to the DNA target that is to be detected in asample. Therefore, when the sample is supplied to the surface of thechip to which the capture probes are attached, if the target DNA ispresent it will bind to the complementary DNA sequence of the captureprobe by the known process of DNA hybridization. The hybridization maypreferably be accelerated by the application of an electric current asis known in the art.

Once the sample has been applied to the chip surface, it is thennecessary to detect any target DNA from the sample that has become boundto the capture probes. To achieve this a solution containingbiotinylated detection probes is added. The detection probes include DNAsequences that are complementary to the target DNA and thus will bind toany target DNA that has previously been caught by the capture probes. Toenable the caught target DNA to be visually detected, gold nanoparticlescoated with streptavidin are added and because of the affinity ofstreptavidin with biotin the gold nanoparticles will become attached tothe detection probes. The gold nanoparticles themselves are too small tobe seen clearly, but a solution containing silver ions may then be addedwhich will be reduced on the surface of the streptavidin-coated goldnanoparticles to faun a silver layer, which is visible as a darkdeposit.

To reduce the background signal the chip may preferably be treated withsalmon sperm DNA. As an alternative to salmon sperm DNA, albumin, orother proteins, or Ficoll, may be used.

The visibility of the silver deposits may be further enhanced by the useof a fixative solution, such as sodium thiosulfate. Conventional imagingequipment and techniques may then be used to detect dark deposits in thereaction cell, which would result from the presence of the target DNA inthe sample.

EXAMPLE

The following example is of a protocol for detecting β-actin DNA in asample. The hybridization steps are electronically assisted, preferablyby pulse hybridization using applied pulses, though continuously appliedcurrent is also possible. Pulse hybridization, however, limits damage tothe chip and results in a higher signal. Typical hybridizationconditions include for the hybridization of the sample DNA to thecapture probes the application of 10 second pulses (13 microamps)followed by a 3 second pause repeated for 8 minutes (i.e. a total of 48pulses. For the hybridization of the detection probe to the capturedsample DNA, the same conditions may be used, but for only 3 minutes(i.e. 18 pulses).

-   -   1. Anti-DIG is diluted 100-fold with 1×PBS (phosphate-buffered        saline), pH 7.4.    -   2. 50 μl of the diluted anti-DIG is added onto an alumina-coated        chip and is incubated at 41° C. for 2 hours.    -   3. The anti-DIG solution is discarded and the chip is washed 3×        with 80 μl 1×PBS, pH 7.4 (pipette up and down during every        wash).    -   4. 1 μl 10 μM of the DIG-labeled capture probes together with        0.2 μg salmon sperm DNA in 50 μl 1×PBS is added onto the chip        and incubated at 41° C. for 30 minutes. The salmon sperm DNA is        firstly denatured by heating to 95° C. to form single strands        and is then mixed with the DIG-labeled capture probes.    -   5. The DIG-labeled capture probe solution and salmon sperm DNA        solution are discarded washed three times with 80 μl 1×PBS and        once with 1× SSPE.    -   6. Add 5 μl 11 μM sample of β-actin sequence (a 91 nucleotide        single-stranded target) in 1× SSPE and apply electric pulse        current as described for 8 min. After hybridization, wash the        chip 3× with 0.1×SSPE.    -   7. Add detection probe (1 ul, 10 μM) to the chip. Apply electric        pulse current as described for 3 min. Wash the chip 3× with        0.1×SSPE and once with 1×PBS.    -   8. Streptavidin-coated gold nanoparticles (obtained from Sigma        Chemical Co., Ltd, St. Louis, Mo., USA) are diluted 10-fold with        1×PBS, pH 7.4.    -   9. The diluted streptavidin-coated gold nanoparticles are added        to the chip surface and incubated at 41° C. for 15 minutes.    -   10. The diluted streptavidin-coated gold nanoparticle solution        is discarded and the chip is washed twice with 80 μl 1×PBS, pH        7.4 (pipette up and down during every wash) and 3× with 80 μl        autoclaved milli-Q water (pipette up and down during every        wash).    -   11. A 1:1 mixture of silver enhancer solutions A and B (obtained        from Sigma Chemical Co., Ltd, St. Louis, Mo., USA) is prepared        just before use and the following steps are performed in a        darkroom. 50 μl of the silver enhancer solution is added onto        the chip and incubated at room temperature for 5 minutes.    -   12. The silver enhancer solution is discarded and the chip        washed once with autoclaved milli-Q water before 50 μl of 2%        sodium thiosulfate is added onto the chip. Pipette up and down        once to remove background and to fix the color.    -   13. Discard the sodium thiosulfate solution after 2 min        incubation and washed with 50 μl milli-Q water before 50 μl        autoclaved milli-Q water is added onto the chip.    -   14. Observe the appearance of dark spots on the chip with an        optical signal detection system.        Experimental Results

The following experimental results shown in FIGS. 2 to 5 were obtainedusing the above hybridization protocol with certain parameters beingvaried as will be understood from the following. In all cases, the basicDNA detection scheme is the following: an aluminum oxide attachmentlayer provided with anti-DIG, DIG-labeled capture probes having a DNAfragment complementary to a single-stranded nucleic acid target,biotin-labeled detection probes for binding to captured targets, andstreptavidin-coated gold nanoparticles with silver enhancement forvisualizing detected target DNA.

FIGS. 2( a)-(d) illustrate the effectiveness of the DIG-labeled captureprobe in the detection of the DNA target. FIGS. 2( a) and (c) both showthe chip before hybridization. The reaction cells are white because nosilver deposits have been formed. FIGS. 2( b) and (d) show the chipafter hybridization with the DIG-labeled capture probe being present inFIG. 2( b) but absent in FIG. 2( d). The reaction cells are seen to bedarker in FIG. 2( b) than in FIG. 2( d) showing the deposit of silver.

FIGS. 3( a)-(d) show that darkened silver deposits are only formed inthe reaction cells in the presence of the target DNA. Similar to FIG. 2,FIGS. 3( a) and (c) show the chip before hybridization, while FIGS. 3(b) and (d) show the chip after hybridization with the target DNA beingpresent in FIG. 3( b) only and not FIG. 3( d). Again it can be seen thatdark deposits of silver are fowled in the reaction cells only in thecase of FIG. 3( b) where the target DNA is present.

FIGS. 4( a)-(d) further demonstrate the effectiveness of the presentinvention by demonstrating that the silver deposits become darker withincreasing target DNA concentration and thus that the optical signalincreases in proportion to the target DNA concentration. FIGS. 4( a)-(c)show the chip after hybridization and detection with (a) undilutedtarget DNA, (b) target DNA diluted 5-fold, and (c) target DNA diluted10-fold. It will be observed that the reaction cells in FIG. 4( a) aredarker than those in FIG. 4( b) which in turn are darker than those inFIG. 4( c). FIG. 4( d) for comparison shows the chip with no target DNApresent and thus white reaction cells with no silver deposited.

Finally, FIG. 5 shows that the intensity of the optical signal varieswith the concentration of the DIG-capture probe. In FIG. 5 theDIG-capture probe concentration varies decreases from left to right andthe reaction cells become correspondingly lighter as less silver isdeposited.

1. Apparatus for detecting target DNA in a biological sample, comprising: (a) a substrate formed with at least one reaction cell, the reaction cell being free of a gel or agarose layer, and wherein said reaction cell includes an aluminum oxide surface for the attachment of DNA capture probes, and at least one of (b) a first reagent comprising capture probes including a DNA fragment that is complementary to said target DNA, (c) a buffer solution for receiving said sample, (d) a second reagent comprising detection probes including a DNA fragment complementary to said target DNA (e) a solution of streptavidin-coated gold nanoparticles, and (f) a solution containing silver ions.
 2. Apparatus as claimed in claim 1 wherein said aluminum oxide surface is formed by oxidation of an aluminum layer.
 3. Apparatus as claimed in claim 1 wherein said aluminum oxide surface is formed by a sputtering technique.
 4. Apparatus as claimed in claim 1 wherein said aluminum oxide surface is coated with a first compound to act as an attachment layer for the capture probe.
 5. Apparatus as claimed in claim 4 wherein said apparatus further comprises capture probes attached to said aluminum oxide surface, and wherein said capture probes are labeled with a second compound which can attach to the said first protein.
 6. Apparatus as claimed in claim 5 wherein said first and second compounds comprise an antibody-protein pair.
 7. Apparatus as claimed in claim 6 wherein said first compound comprises anti-digoxigenin antibodies, and wherein said second compound comprises digoxigenin.
 8. Apparatus as claimed in claim 5 wherein said first and second compounds comprise streptavidin and biotin, or vice versa.
 9. Apparatus as claimed in claim 1 wherein said aluminum oxide surface is coated with a first molecule to act as an attachment layer for the capture probe.
 10. Apparatus as claimed in claim 9 wherein said apparatus further comprises capture probes attached to said aluminum oxide surface, and wherein said capture probes are labeled with a second molecule having an affinity with said first molecule.
 11. Apparatus as claimed in claim 1 wherein said aluminum oxide layer has a thickness of at least 50 Å.
 12. Apparatus as claimed in claim 1 wherein said aluminum oxide surface is coated with a reagent to reduce the background signal in a detection process.
 13. Apparatus as claimed in claim 12 wherein said reagent comprises salmon sperm DNA.
 14. Apparatus as claimed in claim 12 wherein said reagent comprises albumin, or Ficoll. 